PROJECT SUMMARY/ABSTRACT Novel approaches to reduce infarct size (IS) have stalled in translation. Given that clinical treatment of acute myocardial infarction (AMI) is dominated by a rush to open the infarct-related artery, adjunctive therapies which work after reperfusion are highly desirable. Here I propose to investigate the mechanism of cellular postconditioning: cell therapy delivered 20 mins post-reperfusion can reduce the extent of lethal injury, improve functional recovery, and attenuate the progression toward heart failure (HF). The timing is compatible with standard clinical practice in that the decision to treat can be delayed until after the artery has been opened, if an efficacious off-the-shelf product is available. Allogeneic cardiosphere-derived cells (CDCs) are available for immediate use and are in phase 2 clinical testing for chronic MI. Preliminary data from rats show that CDCs, and their secreted exosomes (CDCexo), are cardioprotective when given with a reasonable delay after reflow in AMI. I examined 48 hr and 2 wk endpoints to focus on the acute and chronic benefits of cardioprotection, respectively. To determine the optimal treatment paradigm, I varied systematically the interval between reperfusion and delivery of CDCs or CDCexo. Twenty mins after a 45-min ischemic episode, I saw the greatest decreases of IS, and the best improvements in cardiac function. In published work, I found that macrophages (M?) are essential effectors of cellular postconditioning; their depletion abrogates cellular postconditioning. Conversely, adoptive transfer of CDC- or CDCexo-primed M? is cardioprotective. The major mechanistic objective here is to understand how exosome-mediated changes in M? lead to acute cardioprotection (days after MI) and long-term protection against HF (weeks after MI). Specifically, I will test the concept that CDCexo-primed M? exhibit enhanced efferocytosis (the ability to scavenge toxic cellular debris), thereby improving recovery and preventing progression toward HF. The focus here is on cardioprotection (prevention of cardiomyocyte death), rather than regeneration, and the mechanism of CDC- and CDCexo-mediated enhanced efferocytosis. Mice will be used for all our mechanistic studies. The role of efferocytosis will be probed both by novel in vitro co-culture assays of macrophages, neutrophils, and stressed cardiomyocytes, as well as by in vivo experiments in transgenic mice with MI to quantify and determine the mechanism of CDCexo-mediated efferocytosis. This work has the potential to elucidate the cardioprotective mechanisms of cell therapy that prevent progression to HF.